CN101550406A - Method for preparing multipotential stem cells, reagent kit and application - Google Patents

Method for preparing multipotential stem cells, reagent kit and application Download PDF

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CN101550406A
CN101550406A CNA2008100918412A CN200810091841A CN101550406A CN 101550406 A CN101550406 A CN 101550406A CN A2008100918412 A CNA2008100918412 A CN A2008100918412A CN 200810091841 A CN200810091841 A CN 200810091841A CN 101550406 A CN101550406 A CN 101550406A
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inducible factor
dna
myc
oct4
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CN101550406B (en
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邓宏魁
赵扬
尹晓磊
张蔷
朱芳芳
刘海松
杨炜烽
向晨罡
秦汉
曲秀霞
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Boke Ltd
Peking University
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Abstract

The invention provides a method for efficiently inducing multipotential stem cells, six inducible factors such as Oct4 (POU5f1), Sox2, c-Myc, Klf4, UFT1, Rex1 (ZFP42), and the like and p53 gene inhibitor are induced into the cells by utilizing a slow virus carrier, thereby the method can efficiently induce and produce the multipotential stem cells.

Description

The method for preparing pluripotent stem cell, test kit and purposes
Technical field
The present invention relates to a kind of method of inducing pluripotent stem cell, test kit and purposes.
Background technology
(1) characteristics of stem cell versatility (pluripotency):
Embryonic stem cell is because have to the differentiation capability of three germinal layer cells and be called as " pluripotency " stem cell, and in addition, embryoma (EC) cell and embryonic germ (EG) cell all have and the similar versatility of embryonic stem cell.The pluripotent stem cell common characteristic is: the cell caryoplasm is bigger frequently, and (human embryo stem cell is more flat, and mouse embryo stem cell comparatively swells for the growth of clone's sample, cell boundary is not obvious), have the AP enzymic activity, express SSEA4 specifically, TRA1-60, surface markers such as TRA1-81 (mouse embryo stem cell is also expressed SSEA1 specifically, and human embryo stem cell is not expressed SSEA1), and Oct4, Nanog, Sox2, rex1 (ZFP42), GDF3, lin28, mbd2, significant gene such as TEGF1.In addition, can also be under condition of suspension culture, form embryo's corpusculum (Embryonidbodies) structure, concurrent being conigenous sent out differentiation, adherent culture after EB forms 6-9 days, inwardly can detect, in, the cell of outer three germinal layers differentiation.
(2) research of inducing pluripotent stem cells (IPS cell)
In August, 2006, stretch (Yamanaka) laboratory of covering in regeneration medical courses in general institute of the Kyoto Univ Japan professor mountain and at first announce in the inoblast of mouse, to import 4 gene (Oct4, Sox2, c-Myc and KLF4) successfully it to be induced and become totipotent stem cell, its character and embryonic stem cell are similar. 1Thereby disclosed first by transgenosis and can in the cell of differentiation, set up totipotency.This is a huge impact to for many years biology idea, and is expected to make based on the therapeutic cloning of stem cells technology and thoroughly breaks away from the predicament in ovum source and destroy the ethics dispute that the embryo causes.Use at present this artificial stem cell to carry out the cell therapy patient and remain in safety latent loyally, but it may be applied to research work such as the making of disease model and new drug development in the near future.This achievement has been accelerated the research steps of regenerative medicine, has epoch making significance.
In this experiment, people such as Yamanaka have at first chosen with the mouse embryo stem cell self and have kept 24 relevant genes of versatility, respectively they are cloned into retroviral vector, embryo fibroblast is carried out cotransfection, after the screening of carrying out based on the Fbx15 reporter, they have observed the formation of the cell colony of ES-like.
Through further analysis to these 24 genes, they find, Oct4 only transduces, Sox2, c-Myc, KLF4 just can make embryo fibroblast be transformed into inductive pluripotent stem cell (IPS cell-Induced Pluripotent Stem Cells is commonly called as " inductive pluripotent stem cell ") fully.This IPS cell has normal caryogram, express the molecular marker of similar embryonic stem cell, can external be induced to differentiate into interior, in, the cell of the eventually end differentiation of outer three germinal layers, can in nude mouse, form teratoma, in teratoma, contain interior, in, the noble cells of outer three germinal layers.In addition, the same with embryonic stem cell, the IPS cell detects hypomethylation and the high acetylize of H3 on Nanog and Oct4 promoter.Yet, adopt the Fbx15 gene not have complete totipotency as the cell that reporter screens, as: dryness genes such as endogenous Oct4 still do not have normally to start expression, can not make gomphosis mouse etc. by the method for blastaea injection.
After this, in May, 2007, the statement of on the Nature magazine, publishing an article simultaneously of Yamanaka laboratory and Jaenisch laboratory 2,3They adopt new reporter gene (based on the promoter of Oct4 or nanog) to carry out the IPS cell that the resistance screening can produce complete reprogrammed, new IPS cell can make native gene restart, and can become gomphosis mouse by blastaea injection preparation, and be incorporated in the reproductive system, produce the offspring mouse that generates by this IPS cell fully.The breadboard experimental result of Jaenisch shows, this IPS cell even prepare normotrophic mice embryonic by the chimeric technology of tetraploid embryo.In addition, the modification that methylates of the Oct4 promotor of new IPS cell and Nanog promotor is also almost wiped fully.
Simultaneously, cooperate to publish an article on Cell stem cells with the Plath laboratory in the Hochedlinger laboratory, verified the effect of the complete reprogrammed of IPS cell with abundant more means. 4For example, merge the ability that noble cells is dedifferented, the x chromosome inactivation phenomenon of female cell, the spectrum or the like that methylates of H3K4 and H3K27 with noble cells.4 factors induce noble cells to become the fact of totipotency stem cell by full confirmation thus!
In August, 2007, the Jaenisch laboratory is delivered new article again and is shown: without any need for the screening system, only judge by form equally to obtain reprogrammed totipotent cell system completely. 5If target cell is integrated with the reporter of GFP, can see the startup of endogenous Oct4, but the time of its generation is later, about about 20-30 days, just can observe greatly.Show that also the time of length goes to take place gradually the transformation of reprogrammed to the IPS cell to one of target cell needs.In September in the same year, also publish an article on Cell stem cell magazine and claim and need not any screening system can finish inducing of IPS cell in the Ramalho-Santos laboratory. 6
In addition, also there are some articles that four factors have been carried out further analysis, for example, in the article of above-mentioned Cellstem cell magazine, the author adopts N-myc to replace c-Myc to finish reprogrammed, and also publishing an article on Cell stem cell and NatureBiotechnology respectively and claim and do not use c-Myc in Yamanaka laboratory and Jaenisch laboratory, and only uses Oct4, Sox2 is the same with KLF4 can to obtain the IPS cell 7Yamanaka laboratory even all attempted replacement for other each factor as successfully Sox2 being changed into Sox1 and Sox3, successfully changes Klf4 into Klf2 and Klf5, and has changed c-Myc into L-myc and N-myc.Yet, do not adopt after the c-Myc, induce the efficient of IPS cell to reduce greatly, be not can repeat at every turn yet, prove that c-Myc has still played very crucial effect for reprogramming.
Although induce the technical study development of IPS cell rapid, induce the analysis of IPS cell mechanism but to develop comparatively slow, Yamanaka once proposed model in the document of first piece of IPS cell: the chromosome structure by c-Myc opens noble cells makes it to be easier to be transformed; And KLF4 passes through the P53 signal path with the mutual balance of c-Myc, suppresses the apoptosis that is brought by c-Myc; And Oct4 and Sox2 become a pair of albumen composition, start a series of and the totipotency genes involved, and suppress many and the differentiation and development Expression of Related Genes, thereby make cell be transformed into totipotent transition.
Deliver two pieces of articles respectively on the Cell stem cell magazine in after this, in February, 2008 and in March, 2008 8,9Disclosed the change procedure of inducing the IPS reprogrammed.Wherein, the Jeanisch laboratory utilizes the startup of endogenous dryness genes such as expression, Nanog and Oct4 of four kinds of active appearance of sign: AP, the ssea1 of embryonic stem cell, and the process of inducing of IPS is divided into 3 stages.About about 30 days, wherein the AP activity occurs the induction time of IPS at first greatly, increases in time, and AP active cells ratio increases to more than 80% gradually; Ssea1 began to express about the 9th day, when iPS occurs, had only the cell expressing ssea1 about 15%; Nanog and Oct4 occurred about the 16th day, and ratio is extremely low.Meanwhile, the breadboard work of Hochedlinger also is divided into the process of inducing of iPS the silence of Thy1 similarly, the expression of SSEA-1, the silence of foreign gene, endogenous dryness expression of gene four-stage.These results provide good basis for further furtheing investigate molecular mechanism.Also provide interim index for IPS reprogrammed Study on Technology.
In addition, Yamanaka publishes an article in the Science magazine in February, 2008, has reported and has used new cell type---liver cell of mouse and the process that gastric cells has successfully realized inducing IPS cell reprogrammed. 10What is interesting is, induce the IPS cell still less for the quantity demand of gene integration with liver cell, and littler to the demand of c-Myc gene, compare with the mouse skin inoblast, the trend that easier quilt is induced becomes the IPS cell is arranged.In addition, the classical experimental system that also uses cell line to follow the trail of in breadboard this paper of Yamanaka has been followed the trail of and has been expressed albuminous cell in the former foster liver cell of being commissioned to train, and finds that the most IPS cells that induce at last all derive from the albuminous liver cell of expression.Although whether express albuminous cell must be that the hepatic parenchymal cells that breaks up fully is still waiting discussion, but this experimental result tentatively to becoming somatic reprogrammed ability to carry out more deep judgement and exploration, induces the reprogrammed ability of IPS cell absolutely not only to appear in the inoblast of skin.This provides more reliable foundation for the application of IPS cell technology.
On November 21st, 2007, the complete mouse IPS cell of reprogrammed build be after time of half a year only, just publish an article on Cell and Science magazine respectively in Japanese Yamanaka laboratory and U.S. Thomson laboratory 11, announcing successfully to pass through transgenic method, the human skin cell is induced becomes totipotent IPS cell! Ironically, once adopted 4 factor Oct4 in the work of inducing mouse IPS, Sox-2 have still been used in the Yamanaka laboratory, c-Myc, Klf4, simultaneously, its retroviral acceptor of also having transduceed enters cell with auxiliary retroviral vector, improves gene transfer efficient.New screening has then been carried out to the gene that the embryonic stem cell enrichment is expressed in the Thomson laboratory, and has found the new combinations of factors of inducing IPS: Oct4, Sox2, and Nanog and Lin-28, and use lentiviral vectors to carry out gene transfer.
Only after 1 month, in December, 2007, the U.S. publishes an article on the Nature magazine again in George Daley laboratory, and successfully people's dermal fibroblast being reversed becomes totipotent IPS cell 12After 2 months, in February, 2008, the U.S. has also reported in the Plath laboratory successfully and has induced people IPS cell with the human body inoblast. 13Different is, in the breadboard work of George Daley, must add TERT and two genes that cause cell transformation of SV40T simultaneously when inducing adult's IPS, and the Plath laboratory has then increased a new gene Nanog and lured imported IPS cell.
Summary of the invention
The invention provides a kind of preparation and induce the method for pluripotent stem cell.This method is by providing the inducible factor of particular combinations in the cell of differentiation, induce and produce IPS cell-inductive pluripotent stem cell (Induced Pluripotent Stem Cells, be commonly called as " omnipotent cell "), wherein employed inducible factor relates to Oct4 (POU5f1), Sox2, c-Myc, Klf4, UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition.
On the one hand, the present invention relates to inducible factor UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a is used to the purposes of inducing pluripotent stem cell to produce, and is used to promote induce the purposes and the promotion of the efficient of the factor that produces multipotent stem cells to induce the efficient that produces multipotent stem cells.
Another aspect the present invention relates to the method that pluripotent stem cell is induced in a kind of preparation, and comprising in the cell of differentiation provides inducible factor, described inducible factor comprises Oct4 (POU5f1), Sox2, c-Myc and Klf4, and UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a.Preferably, described inducible factor does not comprise Oct4 (POU5f1), Sox2, a kind of among c-Myc and the Klf4.More preferably, when being UTF1, inducible factor do not comprise Oct4; When being p53si, inducible factor do not comprise KLF4.Still more preferably, described inducible factor does not comprise c-Myc.
In another preferred embodiment, the cell of described differentiation is an inoblast, liver cell, gastric cells, keratinocyte or blood cell.
In another preferred embodiment, the cell of described differentiation derives from Mammals.
In another preferred embodiment, described Mammals is people, mouse or primate.
In another preferred embodiment, described inducible factor is a dna form, mRNA form or protein form.
The p53 expression of gene is the one of the main reasons that causes people and spontaneous differentiation of mouse embryo stem cell and spontaneous apoptosis 14,15In addition, the albumen of p53 genetic expression also can be combined on the special open gene promotor of embryonic stem cells such as Nanog and Oct4, suppresses the expression of these important gene relevant with totipotency.Therefore, suppress the inhibitor of p53 gene transcription product or inhibition P53 function, be expected to promote the totipotency of cell, and suppress differentiation.This can or utilize the small molecules of special inhibition P53 function to suppress the p53 gene by for example synthetic siRNA (intervening rna) fragment, thereby by realizing purpose of the present invention in conjunction with other inducible factor.The present invention has exemplarily proved by experiment and has utilized synthetic p53 to interfere fragment p53si successfully to obtain the IPS cell in conjunction with other inducible factor.Instruction based on specification sheets, those of ordinary skill in the art can determine that fully (as positive-sense strand is 5 ' GACUCCAGUGGUAAUCUACdtdt3 ' to other synthetic intervening rna sequence, SEQ ID NO:54, antisense strand is 5 ' GUAGAUUACCACUGGAGUCdtdt3 ', the intervening rna sequence of SEQ ID NO:55) for example can be by realizing interference in the target cell with Lipofectamine RNA Max (Invitrogen) transient transfection to P53, thus realize purpose of the present invention in conjunction with other inducible factor simultaneously.And those of ordinary skill in the art can determine the pifithrin-α (CALBIOCHEM, article No. 506132) of the micromolecular inhibitor of special inhibition P53 function as the transcriptional activity of inhibition P53 equally based on the instruction of specification sheets 16, suppress P53 and be attached to the go forward side by side pifithrin-miu (CALBIOCHEM, article No. 506155) of an one-step inducing apoptosis of plastosome 17Can in target cell, suppress the effect of P53 equally, realize purpose of the present invention in conjunction with other inducible factor simultaneously.Therefore, based on the instruction of specification sheets, those of ordinary skill in the art can determine that other p53 gene inhibition agent well known in the prior art can be used for the present invention, with other inducible factor in conjunction with obtaining the IPS cell.
In another preferred embodiment, the dna sequence dna of Oct4 (POU5f1) is shown in SEQ IDNO:5, the dna sequence dna of Sox2 is shown in SEQ ID NO:8, the dna sequence dna of c-Myc is shown in SEQ ID NO:14, the dna sequence dna of Klf4 is shown in SEQ ID NO:11, the dna sequence dna of UTF1 is shown in SEQ ID NO:17, the dna sequence dna of Rex1 (ZFP42) is shown in SEQ IDNO:20, agent is selected from the sequence shown in the SEQ ID NO:1 with the p53 gene inhibition, sequence shown in the SEQ IDNO:54, pifithrin-α, or pifithrin-miu.
Those having ordinary skill in the art will appreciate that, when above-mentioned inducible factor is dna form, the technology that is transfected in the cell of differentiation is known in the art, include but not limited to DEAE-dextran method, calcium phosphate method, cationic-liposome method, cationic polymers, Biolistic particle TRANSFER METHOD (particle gun particle bombardment method), microinjection, electroporation and virus-mediated method.The carrier mediated method of preferred virus wherein, described virus vector is preferably lentiviral vectors, retroviral vector, adenovirus carrier etc.Preferably, described slow virus is to be the slow virus (as the PLL3.7 carrier) of representative with I type human immunodeficiency virus (HIV-1), described retrovirus is an example with the PMX carrier, and described adenovirus carrier is to be the duplicate deficit type recombinant adenovirus system of skeleton with Ad5 serotype E 1/E3 defective adenoviral DNA.
Those of ordinary skill in the art is appreciated that equally can be at the above-mentioned inducible factor of vivoexpression, after the acquisition corresponding proteins matter, imports in the cell of differentiation, thereby realizes purpose of the present invention.The technology that protein is imported in the cell is known in the art, includes but not limited to Tat-delivery and correlation technique, Carbon nanotube, Nano-particle, electricity changes (consideration convey dyes), particle gun, ultrasonic+contrast medium, the SLO cell is penetrating, albumen and cell ligand combination. 16
Those of ordinary skill in the art also is appreciated that and the mRNA of above-mentioned inducible factor directly can be imported in the cell of differentiation, it is expressed in cell produce corresponding proteins, thereby realize purpose of the present invention.The technology that mRNA imports in the cell is similar with the technology that protein is imported in the cell, and be known in the art.
Another aspect, the test kit of pluripotent stem cell is induced in a kind of preparation of the present invention, and comprising in the cell of differentiation provides inducible factor, described inducible factor comprises Oct4 (POU5f1), Sox2, c-Myc and Klf4, and UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a.Preferably, described inducible factor does not comprise Oct4 (POU5f1), Sox2, a kind of among c-Myc and the Klf4.More preferably, when being UTF1, inducible factor do not comprise Oct4; When being p53si, inducible factor do not comprise KLF4.Still more preferably, described inducible factor does not comprise c-Myc.
In another preferred embodiment, described inducible factor is a dna form, mRNA form or protein form.
In a preferred embodiment, when described inducible factor was dna form, described test kit also comprised and being used for the virus vector of inducible factor transfection to noble cells.
In another preferred embodiment, described virus vector is selected from lentiviral vectors, retroviral vector or adenovirus carrier.
In another aspect of the present invention, relate to pluripotent stem cell with aforesaid method and test kit preparation.
By additional P53si on the basis of 4 reprogrammed genes having reported, UTF1, three inducible factors of Rex1 or wherein any all can obviously improve reprogramming efficiency, and can produce normal IPS cell.UTF1 even can replace Oct4 in 4 factors of having reported; And P53si can replace the Klf4 in 4 factors of having reported and realize reprogrammed.
The present invention obtains induces pluripotent stem cell, express the significant gene and the surface protein of embryonic stem cell, the same alkaline phosphatase activities with embryonic stem cell with surface, ability with the spontaneous embryo's of being differentiated to form corpusculum (Embryoid body), and can by further differentiation become interior, in, the cell type of outer three germinal layers.With the report unanimity in the mouse is that this process of inducing has experienced several developmental stage: the generation of AP+ cell, the generation of the cell clone of ES-like, the startup of the silence of external source quiding gene and endogenous dryness gene.
The IPS cell that uses this paper inventive method to set up has very high efficient, from the AP+ stage that the IPS cell formation process must pass through, use 7 inducible factors to carry out the IPS cell preparation and can be not enough to effectively to produce under the situation of IPS cell (seeing Table 1) in 4 factors in this laboratory and set up out IPS clone in a large number, and to build than the IPS cell of having reported be that efficient exceeds 1-2 the order of magnitude.Can clearer and more definite P53si by 7 inducible factors are further analyzed, UTF1 and Rex1 have the obvious contribution for reprogramming efficiency.Wherein, UTF1 even can replace Oct4 in 4 factors of having reported; And P53si can replace the Klf4 in 4 factors of having reported and realize reprogrammed.
Description of drawings
Fig. 1 is the structure iron of lentiviral vectors pLL3.7 used herein, a) is slow virus interference vector pLL3.7; B) be slow virus interference vector pLL-IRES-puro.
Fig. 2 is the fluorogram of lentiviral vectors transfection EGFP, a) is the aspect graph of cell under the common visual field; B) show a high proportion of green fluorescence for cell.
Fig. 3 is the AP positive colony of slow virus infection inoblast after 20 days that carries each inducible factor, a) is: become human foreskin fibroblast (HFF), the target cell number is 2 * 10 5, AP+ and similar embryonic stem cell clone number are 181; B) for embryo skin becomes fiber (fsf), the target cell number is 3 * 10 5, AP+ and similar embryonic stem cell clone number are 200; C) for the tire lung becomes fiber (FL), the target cell number is 2 * 10 5, AP+ and similar embryonic stem cell clone number are 152.
Fig. 4 is IPS cell expressing human embryo stem cell surface marker and AP stained positive, wherein schemes a) to be the aspect graph of IPS cell clone; B) be the AP colored graph of IPS cell; C) be nucleus DAPI colored graph; D) be and c with the SSEA4 immunofluorescence figure under the visual field; E) be nucleus DAPI colored graph; F) be and the Nanog immunofluorescence dyeing figure of e with the visual field.
Fig. 5 is the specific gene of IPS cell expressing human embryo stem cell.Wherein, 3AS is the IPS cell of setting up with 7 factor methods (express foreign gene silence, endogenous dryness gene and other dryness marker gene are expressed); 3B is the cell (positive control of exogenous gene expression) in the reprogrammed process; H1 is the embryonic stem cell contrast, the positive control of dryness genetic expression; FSF is the target cell (the subcutaneous inoblast of embryo) that is used to induce 3AS clone, is the negative control of dryness gene and external source quiding gene.GAPDH is the positive control of the RT-PCR experiment of all cells.
Fig. 6 for the IPS cell form embryo's corpusculum (Embryoid Bodies, EB).
Fig. 7 is sent to the differentiation of three germinal layer cells certainly by EB for the IPS cell.Wherein AFP is the sign of entoderm liver cell, and T is the gene of mesoblastema specifically expressing, and nestin is the gene of neurocyte specifically expressing. 11，13
Fig. 8 is the AP+/similar embryonic stem cell clone synoptic diagram of inducing the multipotency STEM CELL FACTOR to obtain.Ordinate zou: per 10 5Clone's number that cell produces, grey post are represented clone's sum, and the black post is represented clone's number of AP strong positive, the white post is represented clone's number of AP strong positive and ES-like, and wherein O represents Oct4, and S represents Sox2, M represents c-Myc, and K represents Klf4, and P represents P53si, U represents Utf1, and R represents Rex1, and 7 is the combination of OSMKPUR7 the factor, 7-O deducts this factor of O in 7 factors, by that analogy, 4P is OSMKP, and 3P is OSMP.
Embodiment
Further to illustrate the present invention by embodiment.Those having ordinary skill in the art will appreciate that the present invention is not limited to described embodiment, and those of ordinary skill in the art can make amendment based on the instruction of specification sheets to embodiment.These modifications are included in the present invention equally by in the defined scope of the present invention of accompanying Claim.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
The structure of embodiment 1. recombinant vectors pLL-p53si
1) .p53 interferes segmental acquisition
Synthetic p53 interferes segmental positive-sense strand and antisense strand according to following sequence:
Positive-sense strand: 5 '-TGACTCCAGTGGTAATCTACTTCAAGAGAGTAGATTACCACTGGAGTCTTTTTTC-3 ' (SEQ ID NO:1)
Antisense strand: 5 '-TCGAGAAAAAAGACTCCAGTGGTAATCTACTCTCTTGAAGTAGATTACCACTGGAG TCA-3 ' (SEQ ID NO:2)
The following annealing in the 1.5ml pipe with positive-sense strand and antisense strand, reaction system 10ul, add respectively: 10 * annealing buffer (100mM Tris-HCl, pH7.5,1M NaCl, 10mM EDTA) 1ul, each 1ul of positive-sense strand and antisense strand (500ng/ul), an amount of sterilized water complements to 10ul, boils postcooling and spends the night, and obtains p53 and interferes fragment.
Slow virus interference vector pLL3.7 (Rubinson and Dillon et al, Nature Genetics, 2003, plasmid map sees on Fig. 1 that specifying information and complete sequence can references Http:// www.sciencegateway.org/protocols/lentivirus/pllmap.html, SEQ IDNO:21) with Hpa I and Xho I at the pLL3.7 plasmid of 37 ℃ of following double digestions through checking order correct, use the fragment of DNA fast purifying/recoverys test kit recovery 7.6kb size of ancient cooking vessel state company.
Use DNA Ligation Kit Ver2.0 (TaKaRa), draw an amount of DNA respectively in the 1.5ml pipe according to 10: 1 the ratio of inserting of DNA/ carrier mol ratio, reaction system 10ul adds respectively: 10 * damping fluid 1ul, 10mM dNTPs 0.2ul, T 4Dna ligase 0.2ul, an amount of sterilized water complements to 10ul, reacts 30min under 16 ℃ of conditions.
Get 5ul connection product and join in the 100ul Top10 competent cell ice bath 30 minutes; Hatched 90 seconds for 42 ℃; Ice bath is 2 minutes again; It is transferred in the 0.5ml LB nutrient solution, cultivated 45 minutes under 37 ℃, 100rpm condition; Draw then on the LB flat board that 50ul evenly coats diameter 10cm, cultivated 18 hours under 37 ℃ of conditions.Normal, finely disseminated 5 the single bacterium colonies of picking form send order-checking to identify at random, obtain recombinant vectors pLL-p53si.
Embodiment 2.PLL-Oct4 (POU5f1), PLL-Sox2, PLL-c-Myc, PLL-Klf4, PLL-UTF1, the structure of PLL-Rex1 (ZFP42) expression vector
1.pLL-IRES-puro the extraction of plasmid
1) with Xba I and Not I double digestion pLL3.7, mend with T4 DNA polymerase then and put down, connect with T4 DNA Ligase, remove U6 promotor and Lox P, obtain pLL3.7-U6-.With Nhe I single endonuclease digestion, reclaim big fragment, and use the alkaline phosphatase dephosphorylation, obtain enzyme and cut carrier;
2) pIRES (Clontech) mends with T4 DNA polymerase then and puts down with EcoR I single endonuclease digestion, connects with T4 DNA Ligase, remove EcoR I site, obtain pIRES-E-, with Nhe I and Xba I double digestion pIRES-E-, reclaim the 700bp fragment, obtain the IRES fragment.
3) IRES fragment and the 1st) enzyme that obtains of step is cut carrier and is connected with T4 DNA Ligase, obtains pLL-IRES-GFP.BamH I and the correct pLL-IRES-GFP of EcoR I double digestion, and reclaim big fragment, obtain enzyme and cut the pLL-IRES carrier;
4) with primer 5 ' GTAGGATCCATGACCGAGTACAAGCCC 3 ' (SEQ IDNO:52) and 5 ' GATGAATTCAGGCACCGGGCTTGCG 3 ' (SEQ ID NO:53), pcr amplification pBabePuro (Search Vectorpedia) obtains the Puro PCR fragment of 800bp.With BamH I and EcoR I double digestion Puro PCR fragment, reclaim, obtain enzyme and cut puro, being connected to the 3rd) enzyme in step cuts the pLL-IRES carrier, obtains pLL-IRES-Puro.
The Wizard Plus SV Minipreps DNA PurificationSystem test kit of using Promega company extracts the pLL-IRES-puro plasmid, and collection of illustrative plates is seen Fig. 1 b).
2.Oct4 (POU5f1), Sox2, c-Myc, Klf4, UTF1, the acquisition of Rex1 (ZFP42) cDNA
Extract the RNA of human embryo stem cell according to ordinary method, amplify Oct4 (POU5f1) by RT-PCR, Sox2, c-Myc, Klf4, UTF1, the cDNA of Rex1 (ZFP42) gene, with its be cloned in the pGEM-T carrier order-checking correct after, under the Xho I+EcoR I double digestion cDNA fragment and be cloned into the CMV promotor of pLL-IRES-puro carrier after.Specific as follows.
1) human embryo stem cell RNA extracts
1. 3.5 millimeters culture dish cultured human embryo tire stem cells are centrifugal in the Eppendorf pipe (nuclease free) of 1.5ml, and (room temperature left standstill 5 minutes for Invitrogen, Cat.No.15596-026) fully homogenate to add 1ml Trizol;
2. add the 0.2ml chloroform, thermal agitation 15s leaves standstill 2min;
3. 4 ℃ centrifugal, 12000g * 15min gets supernatant;
4. add the 0.5ml Virahol, with the mixing gently of liquid in the pipe, room temperature leaves standstill 10min;
5. 4 ℃ centrifugal, 12000g * 10min abandons supernatant;
6. add 1ml 75% ethanol, washing precipitation gently, 4 ℃, 7500g * 5min abandons supernatant;
7. dry, adding 100ul does not have RNase water dissolution (65 ℃ of short molten 10-15min).
The total RNA that extracts is used for reverse transcription immediately or places-80 ℃ of preservations.
After 50 times of RNA dilutions, measure A in ultraviolet spectrophotometer 260/280The OD value.OD 260=0.699; OD 280=0.435; A 260/280=1.607; The concentration of total RNA is: 0.699 * 40ug/ml * 50 (extension rate)=1398ug/ml.
2). cDNA first chain is synthesized in reverse transcription
1. get the total RNA of 0.7ul (being equivalent to the total RNA of 1ug) in 200ul PCR pipe, hatch for 70 ℃ and be placed on ice in 10 minutes.
2. in above-mentioned PCR pipe, add solution, be prepared into the reverse transcription reaction system of 20ul with lower volume:
25mM?MgCl 2 4ul
10×RT?buffer 2ul
DNTP mixture 2ul
RNA enzyme inhibitors 0.5ul
AMV ThermoScript II 0.5ul
Oligo(dT)15 1ul
The water of nuclease free is to final volume 20ul
3. after 42 ℃ of above-mentioned reaction systems are hatched 15 minutes, 95 ℃ of heating 5 minutes, 4 ℃ of insulations placed-20 ℃ of preservations standby after 5 minutes.
3) .PCR each gene cDNA that increases
MRNA sequence according to each gene among the GeneBank designs a pair of primer P1 and P2 respectively, introduces Xho I restriction enzyme site among the primer P1, and primer P2 introduces EcoR I restriction enzyme site.The PCR reaction obtains the cDNA of each gene.(100ul) is as follows for reaction system:
Aseptic deionized water 71ul
10×Pyrobest?buffer?II 10ul
P1(10uM) 2ul
P2(10uM) 2ul
DNTP mixture (each 2.5mM) 8ul
cDNA 5ul
Pyrobest archaeal dna polymerase 2ul
The PCR reaction conditions is as follows:
35 circulations are carried out in following sex change → annealing → extension.
Sex change: 94 ℃, 30sec;
Annealing: 56 ℃, 40sec;
Extend: 72 ℃, 2min.
The open reading frame sequence that obtains of Oct4 primer and PCR wherein
P1：5’AGGATCCGCCACCATGGCGGGACACCTGGC?3’(SEQ?IDNO：3)
P2：5’GCGAATTCATCAGTTTGAATGCATGGGAGG?3’(SEQ?IDNO；4)
The ORF of Oct4: see SEQ ID NO:5
The open reading frame sequence that Sox2 primer and PCR obtain
P1：5’CCTCGAGCCACCATGTACAACATGATGGAG?3’(SEQ?IDNO：6)
P2：5’CGGAATTCATCACATGTGTGAGAGGGGC?3’(SEQ?ID?NO：7)
The ORF of Sox2 sees SEQ ID NO:8
The open reading frame sequence that Klf4 primer and PCR obtain
P1：5’TACTCGAGGCCACCATGGCTGTCAGCGACGC?3’(SEQ?IDNO：9)
P2：5’GGCGAATTCATTAAAAATGCCTCTTCATGTG?3’(SEQ?IDNO：10)
The ORF of Klf4 sees SEQ ID NO:11
The open reading frame sequence that c-Myc primer and PCR obtain
P1：5’ACTCGAGCCACCATGCCCCTCAACGTTAGC?3’(SEQ?IDNO：12)
P2：5’CGGAATTCATTACGCACAAGAGTTCCGTAG?3’(SEQ?IDNO：13)
The ORF of c-Myc sees SEQ ID NO:14)
The open reading frame sequence that Utf1 primer and PCR obtain
P1：5’AACTCGAGCCACCATGCTGCTCCGGCCCCGC?3’(SEQ?IDNO：15)
P2：5’AAGAATTCCACTGGCACGGGTCCCTGAGGA?3’(SEQ?IDNO：16)
The ORF of Utf1 sees SEQ ID NO:17
The open reading frame sequence that Rex1 (ZFP42) primer and PCR obtain
P1：5’AACTCGAGCCACCATGAGCCAGCAACTGAAGAAAC?3’(SEQ?ID?NO：18)
P2：5’TTGATATCCTACTTTCCCTCTTGTTCATTC?3’(SEQ?ID?NO：19)
The ORF of Rex1 (ZFP42) sees SEQ ID NO:20
4) recovery of .PCR product
After the PCR product carried out gel electrophoresis, use DNA fast purifying/recoverys test kit (Beijing ancient cooking vessel state biotech firm, cat.no A014-1) and reclaim big or small separately band.The DNA that obtains is diluted 20 times, measure A down in ultraviolet spectrophotometer 260/280OD.A 260=0.035; A 280=0.022; A 260/280=1.609; DNA concentration is: 0.035 * 50ug/ml * 20=35ng/ul.
5) evaluation of .PCR product
(reaction system is 10ul to the PCR product that reclaims, and is specially PCR product 7.2ul, 10 * PCR damping fluid 1ul after the external polyA of adding reaction, dNTP mixture (2.5mM) 0.8ul, TaqDNA polysaccharase 1.0ul, reaction is 30 minutes under 70 ℃ of conditions), be connected into pGEM-T carrier (Promega).The recombinant plasmid that obtains identifies that through above-mentioned PCR, Xho I and EcoR I double digestion true positive recombinant plasmid delivers the order-checking of Beijing three rich polygala root biotech firms.Sequencing result shows that each gene order of cloning among the pGEMT is entirely true.
6) .pLL-Oct4 (POU5f1), pLL-Sox2, pLL-c-Myc, pLL-Klf4, the structure and the evaluation of pLL-UTF1 and pLL-Rex1 (ZFP42) recombinant vectors
1) enzyme is cut and is reclaimed
1. the enzyme of each gene is cut and is reclaimed
The recombinant plasmid that obtains in the correct step 5) that checks order with Xho I and EcoR I double digestion is to reclaim the cDNA of each gene, 37 ℃ of incubations.After enzyme to be determined is cut fully, stopped reaction.Use the DNA fast purifying/recovery test kit of ancient cooking vessel state company to reclaim the fragment of each gene size.
2. the enzyme of pLL-IRES-puro plasmid is cut and is reclaimed
Use Xho I and 37 ℃ of enzymes of EcoR I (NEB company) to cut the pLL-IRES-puro plasmid.After enzyme to be determined is cut fully, stopped reaction.DNA fast purifying/recovery test kit of use ancient cooking vessel state company reclaims the DNA (6.5kb) after enzyme is cut.
2) ligation
Use DNA Ligation Kit Ver2.0, draw an amount of DNA respectively in the 1.5ml pipe according to 3: 1 the ratio of inserting of DNA/ carrier mol ratio, reaction system 10ul, add respectively: 10 * buffer 1ul, 10mM dNTPs 0.2ul, T4DNA ligase enzyme 0.2ul, an amount of sterilized water complements to 10ul, reacts 30min under 16 ℃ of conditions.
3) transform and identify
As above will connect product according to ordinary method is transformed in the Top10 competent cell.After PCR identifies and order-checking has obtained pLL-Oct4 (POU5f1) respectively, pLL-Sox2, pLL-c-Myc, pLL-Klf4, pLL-UTF1 and pLL-Rex1 (ZFP42) recombinant vectors.
Embodiment 3. human fibroblastss' (target cell) acquisition
1) fresh embryo's prepuce tissues (Fsf), tire lung (FL) and adult prepuce tissues (HFF) 75% ethanol disinfection are also with phosphate buffered saline buffer (PBS) washing;
2) go out subcutis with the eye scissors careful separation and shred;
3) wash several times with PBS again, get small tissue blocks and be inoculated in the culture dish, be positioned at 37 ℃, in 5% CO2gas incubator;
4) (available from Hyclone company, catalog number is SH30022.01B, wherein adds 15% foetal calf serum (FBS) to add the high dextrose culture-medium of DMEM after two hours, 0.1mM beta-mercaptoethanol, 1% non-essential amino acid, 1mM glutaminate, 8 units/ml gentamicin);
5) cultivate 2-3 days, remove can not be adherent tissue block;
6) continue to cultivate 7-9 days, remove remaining tissue block;
7) 0.25% trypsinase and 0.02%EDTA room temperature peptic cell are 5 minutes, with the high dextrose culture-medium neutralization of above-mentioned DMEM, are inoculated in new culture dish at 1: 3;
8) changed liquid in per 2 days after, went down to posterity in per 4 days 1: 3.
Embodiment 4. people induce inducing of pluripotent stem cell
1) preparation of slow virus
The slow virus that this experiment is adopted is the recombinant vectors that has carried above-mentioned each inducible factor that utilizes embodiment 1 and 2 preparations, utilizes available from preparing according to ordinary method in the packaging plasmid pMDLg/pRRE of Addgene company (article No. 12251) pRSV-Rev (article No. 12253) and pMD2.G (article No. 12259).
Concrete operation method is as follows:
EndoFree Plasmid Kit (Qiagen) prepares above-mentioned plasmid in a large number.The plasmid that extracts is frozen in TE with the concentration of 2-4ug/ul.
The ratio of four plasmids is as follows during packing:
Each recombinant vectors of embodiment 1 and 2 preparations: 15ug
pMDLg/pRRE： 5ug
pRSV?REV： 7ug
pVSVg： 5ug
Packaging step:
1. go down to posterity with the DMEM (available from Hyclone company, catalog number is SH30022.01B) that contains 10% foetal calf serum and cultivate HEK293T cell (ATCC, catalog number CRLv-11268), 8x10 6Cell/10cm culture dish, second day observation of cell, cell wants the border clearly demarcated, and non-agglomerate is not piled up, and forms an individual layer.Intracellular particle is few, and it is full that cell seems, three to four projections are arranged, and projection can not be very long.Outside boundary curve slyness then, do not have tiny projection, probably account for 60-80%, can pack;
Change the fresh DMEM substratum that contains 10% foetal calf serum 2. for before the transfection 293T cell;
3. Bao Zhuan liquid is divided into 2 * HBS, water, 2.5M CaCl 2, take out a collection of 1.5ml pipe earlier, add 400ul water earlier, add the 2.5M CaCl of 50 μ l again 2, mixing adds four plasmids more successively, and mixing is adjusted the cumulative volume of water according to the cumulative volume of plasmid, and making final volume is 500ul.Get the 5ml fluidic cell pipe of equal amount, every pipe adds 2 * HBS of 500ul.The speed of mixing solutions in the 1.5ml pipe with one of per second is joined among 2 * HBS of streaming pipe, one of every adding, rolling is even immediately.After the mixing, visible solution is creamy white, and thorough mixing under the back whirlpool concussion 5-6 that closes the lid is to avoid forming very big calcium phosphate precipitation.
4. mixed solution dropwise adds in the corresponding 293T cell; (can see the tiny grain of calcium phosphate precipitation after 10-20 minute) at microscopically
5. arrive the DMEM substratum of changing 10% fresh foetal calf serum after 12-16 hour;
6. (carry the same respectively pMDLg/pRRE of recombinant vectors, pRSV REV, the pVSVg plasmid co-transfection cell of above-mentioned each inducible factor according to the ordinary method transfection, the slow virus of carrying above-mentioned each inducible factor with packing respectively) confirmed quantity and the state of target cell in back 24 hours, the target cell of 10cm culture dish, every ware about 10 are used in every part of transfection 6Individual cell;
7. receive poison after the transfection after 44-48 hour;
Take out 293T supernatant after the transfection earlier (if many 293T cell suspensions are arranged in the supernatant with syringe when 8. receiving poison, also can re-use syringe behind the centrifugal 10min of first 3000rpm), filter (0.45 μ M filter) and remove the 293T cell, obtain the slow virus of carrying above-mentioned each inducible factor respectively thus;
9. 1: 1 polyinfection target cell of venom (being used in combination of venom) and substratum referring to the combinations of factors in the table 1 (venom of each 10cm culture dish results infects the target cell of a 10cm culture dish;
10. infect the DMEM fresh culture of changing 10% foetal calf serum after 8-12 hour
Infect back 48 hours according to ordinary method detection efficiency of infection.As shown in Figure 2, the cell that is had a virus infection of green fluorescent protein accounts for more than 90% of total cellular score.
2) the further inducing culture of slow virus infection cell
The target cell of slow virus infection continues to cultivate 5 days in the DMEM+10% foetal calf serum.Infect back the 7th day with 5x10 4The density of cell/10cm culture dish is inoculated on feeder layer (feeder) cell.
The acquisition of feeder layer (feeder) cell:
1. (mouse embryonicfibroblast MEF), discards MEF substratum (the DMEM substratum adds 10% foetal calf serum), adds the ametycin working fluid that contains the 10ug/mL ametycin to get the good adherent mouse embryo fibroblasts of growth conditions;
2. cultivated 3 hours down at 37 ℃, handle the culture dish that will inoculate the MEF cell with 0.1% gelatin between incubation period, room temperature is placed (or 37 ℃ of placements are more than 30 minutes) more than 2 hours, gets final product with before sopping up gelatin solution;
3. take out the MEF cell, discard and contain the ametycin working fluid, wash 5 times with PBS, so that thoroughly wash remaining mitomycin off;
4. add pancreas enzyme-EDTA (U.S. Gibco company) and digest, use MEF substratum termination reaction then;
5. 1000 rev/mins of kinds are centrifugal 5 minutes, abandon supernatant, with MEF substratum re-suspended cell precipitation counting also;
6. according to 1.6 * 10 5The density of individual cell/3.5cm culture dish will place 37 ℃ of incubators to cultivate 12-24 hour through MEF cell inoculation that above-mentioned steps is handled to the culture dish that is coated with 0.1% gelatin, obtain being used for the feeder layer of cultivator embryonic stem cell.
Target cell reaches second day substitution embryonic stem cell substratum after the feeder layer cells.Fill a prescription following 20% serum substitute (Knock-out SerumReplacement, KSR, Gibico), the 1mM glutamine, 0.1mM beta-mercaptoethanol, 1% non-essential amino acid (U.S. Gibco company), 4ng/mL Prostatropin (bFGF) is used ddH 2O is settled to 1000mL.
Continue to cultivate, changed liquid in per two days.Infect the expression of the alkaline phosphatase (AP) of the back 20 days infected cells of available Alkaline PhosphataseDetection Kit (Chemicon) detection.The result is illustrated in figure 3 as the AP positive colony of each slow virus infection inoblast of carrying inducible factor after 20 days, a) is: become human foreskin fibroblast (HFF), the target cell number is 2 * 10 5, AP+ and similar embryonic stem cell clone number are 181; B) for embryo skin becomes fiber (fsf), the target cell number is 3 * 10 5, AP+ and similar embryonic stem cell clone number are 200; C) for the tire lung becomes fiber (FL), the target cell number is 2 * 10 5, AP+ and similar embryonic stem cell clone number are 152.
Clone's (tentatively be called the IPS cell, promptly the inductive pluripotent stem cell sees that Fig. 4 a)) of picking AP expression positive (being AP+) and the similar human embryo stem cell of form reaches on the new feeder layer cells from remaining cell.Cultivate according to the conventional cultural method continuation of human embryo stem cell subsequently and go down to posterity:
1) adding or 1mg/mL collagenase IV (Gibco) place 37 ℃ of incubators to hatch 10-15 minute, take out cell then and observe under phase microscope, if crimping appears in the clone edge, then can stop digestion; Otherwise put back to incubator, prolong digestion time, but will take out observation at any time, to prevent excessively to cause the clone to come off because of digesting;
4) after digestion finishes, sop up Dispase or collagenase IV, after usefulness PBS and DMEM/F12 substratum (Gibco, article No. 11330-032) are washed one time respectively, add an amount of DMEM/F12 (2mL/3.5cm culture dish);
5) gently cell clone is scraped from the culture dish bottom with aseptic straight peen or elbow glass dropper, and be transferred in the aseptic 15mL tapered bottom centrifuge tube,, make cell clone become the comparatively little cell mass of homogeneous of size with dropper pressure-vaccum several times leniently;
6) the centrifugal 3-4 of 1000rpm minute, carefully sop up supernatant, inhale the fresh resuspended precipitation of human embryonic stem cell medium with the glass dropper;
7) take out the MEF feeder layer cells of above-mentioned acquisition, give a baby a bath on the third day after its birth time, the little agglomerate of above-mentioned cell is inoculated on the MEF feeder layer cells, place 37 ℃ of cell culture incubators to cultivate replaceable fresh HESM substratum behind the cell attachment 12-24 hour with PBS.Change a subculture every day, went down to posterity once in 5-7 days usually.If one of following situation Shi Zexu occurring in time goes down to posterity: reached for two weeks the storage period of (1) MEF feeder layer; (2) too densification or area are excessive for cell clone; (3) tangible spontaneous differentiation appears in cell.
Embodiment 5. induces the totipotent evaluation of pluripotent stem cell
1). alkaline phosphatase (AP) dyeing and immunohistochemical staining detect the dryness expression of gene
The existence of alkaline phosphatase is the important indicator that embryonic stem cell keeps undifferentiated state, and whether the existence by detecting it, can further judge embryonic stem cell.Contain abundant alkaline phosphatase (AP) in the embryonic stem cell, the embryonic stem cell AP of differentiation is weak positive or negative.
According to the explanation of manufacturers, detect the expression of the alkaline phosphatase (AP) of inductive pluripotent stem cell with Alkaline Phosphatase Detection Kit (Chemicon).The result as Fig. 4 a) shown in, inducing pluripotent stem cells 3AS clone has the alkaline phosphatase activities the same with embryonic stem cell.
For further detect with the cell of induction method acquisition of the present invention whether really as keeping of being showed on the form undifferentiated state of cell, use the painted method of cellular immunofluorescence further to detect the IPS cell endogenous dryness gene Nanog in 9 generations and the expression of SSEA4 gene (the SSEA4 gene is the unique tag on embryonic stem cell surface), concrete grammar may further comprise the steps:
1) takes out cell, abandon substratum, wash twice with PBS;
2) the Paraformaldehyde 96 room temperature of adding 4% is fixed 15 minutes, or adds the anhydrous methanol room temperature fixedly 5-10 minute;
3) give a baby a bath on the third day after its birth time with PBS, every all over 5 minutes;
4) change thoroughly with PBST (the PBS solution that contains 0.2%Triton X100 (volume percent)) solution, room temperature was placed 10 minutes;
5) wash one time 5 minutes with PBS;
6) add the PBST that contains 2-3% lowlenthal serum or horse serum, room temperature sealing 30-60 minute;
7) abandon confining liquid, add one anti-(rabbit is anti--Oct4, mouse-anti-Tra-1-81, U.S. Chemicon company) (by 1: 50-200 dilutes with confining liquid (PBST that contains 2-3% lowlenthal serum or horse serum)), 4 ℃ of placements 12-24 hour (or 37 ℃ hatched 2 hours);
8) give a baby a bath on the third day after its birth time with PBS, every all over 5 minutes;
9) add two anti-(rhodamine mark goat is anti--rabbit igg, rhodamine mark rabbit be anti--mouse IgG, mountain Bioisystech Co., Ltd in the China) (by 1: the 50-150 dilution proportion (takes by weighing 0.1g bovine serum albumin (bovine serum albumin in two anti-diluents, BSA), be dissolved among the PBS of 100mL, be 0.1% BSA solution)) in, 37 ℃ of lucifuges were placed 1 hour;
10) give a baby a bath on the third day after its birth time with PBS, every all over 5 minutes;
11) adding final concentration is the DAPI solution (U.S. Roche company) of 1mg/mL, and room temperature was placed 5 minutes;
12) give a baby a bath on the third day after its birth time with PBS, every all over 5 minutes;
13) add 500ul PBS (or PBS: glycerine (1: 1)), under fluorescent microscope, observe and take pictures.
The result is as Fig. 4 c)-f) shown in.The IPS cell that derives with our method has the same expression that can detect surface marker SSEA4 with embryonic stem cell, and the expression of the crucial endogenous transcription factor nanog of embryonic stem cell.
2) .RT-PCR detects the dryness expression of gene
The dryness expression of gene situation of inductive IPS cell is carried out RT-PCR detect, concrete grammar may further comprise the steps:
1) use the Trizol method to extract the RNA of culturing cell;
2) reverse transcription of RNA: use the reverse transcription test kit of Promega company and by specification to operate synthetic its cDNA of reverse transcription;
3) cDNA of polymerase chain reaction (polymerase chain reaction, PCR): with step 2) acquisition is a template, carries out PCR and detect under the guiding of upstream primer P3, downstream primer P4 (seeing below).The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 40sec then, 53-62 ℃ of 40sec, 72 ℃ of 30-60sec, totally 35 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 2% agarose gel electrophoresis detect.Detected result is seen Fig. 5, and wherein H1 represents the embryonic stem cell positive control; 3AS represents the IPS cell (its endogenous gene expression with the embryonic stem cell positive control is consistent for foreign gene silence, endogenous dryness genetic expression) set up with above-mentioned 7 kinds of inducible factors; 3B represents the cell of noble cells in IPS cell reprogrammed process (foreign gene strongly expressed, native gene is not activated, and proves successfully the foreign gene transfection in cell); FSF represents to induce the target cell (the subcutaneous inoblast of embryo) (seeing description of drawings for details) of 3AS clone
Detect with primer as follows:
Native gene:
Oct4:5 ' GAACCGAGTGAGAGGCAACC 3 ' (SEQ ID NO:22) and 5 ' ATCCCAAAAACCCTGGCACA 3 ' (SEQ ID NO:23)
Sox2:5 ' ATGGGTTCGGTGGTCAAGTC 3 ' (SEQ ID NO:24) and 5 ' CCCTCCCATTTCCCTCGTTT 3 ' (SEQ ID NO:25)
Nanog:5 ' TGGAACAGTCCCTTCTATAA 3 ' (SEQ ID NO:26) and 5 ' CTGATTAGGCTCCAACCATA 3 ' (SEQ ID NO:27)
Foreign gene:
Klf4:5 ' ACCACTGTGACTGGGACG 3 ' (SEQ ID NO:28) and 5 ' GCAGCGTATCCACATAGCGT 3 ' (SEQ ID NO:29)
Myc:5 ' TACATCCTGTCCGTCCAAGC3 ' (SEQ ID NO:30) and 5 ' GCAGCGTATCCACATAGCGT 3 ' (SEQ ID NO:31)
Rex1:5 ' TCATTCATGGTCCCCGAGA 3 ' (SEQ ID NO:32) and 5 ' GCAGCGTATCCACATAGCGT 3 ' (SEQ ID NO:33)
Utf1:5 ' GACCAGCTGCTGACCTTGA (SEQ ID NO:34) 3 ' and 5 ' GCAGCGTATCCACATAGCGT 3 ' (SEQ ID NO:35)
The primer of the peculiar gene of embryonic stem cell is used for increasing:
P53:5 ' CAGCCAAGTCTGTGACTTGCACGTAC 3 ' (SEQ ID NO:36) and 5 ' CTATGTCGAAAAGTGTTTCTGTCATC 3 ' (SEQ ID NO:37)
Lin28:5 ' GGGCATCTGTAAGTGGTT 3 ' (SEQ ID NO:38) and 5 ' GTAGGGCTGTGGATTTCT 3 ' (SEQ ID NO:39)
Gdf3:5 ' CCCGAGACTTATGCTACG 3 ' (SEQ ID NO:40) and 5 ' TCCAGGAATAACCCGAAA 3 ' (SEQ ID NO; 41)
Hesx1:5 ' AAACCCTCAACTTGCTCC 3 ' (SEQ ID NO:42) and 5 ' TTGGTCTTCGGCCTCTAT 3 ' (SEQ ID NO; 43)
Tdgf1:5 ' TCAGGAATTTGCTCGTCC 3 ' (SEQ ID NO; 44) and 5 ' CTTGGGCAGCCAGGTGT 3 ' (SEQ ID NO:45)
Mbd2:5 ' ATCTGGGCTAAGTGCTGG 3 ' (SEQ ID NO:46) and 5 ' AAGCTGGGTCTTGGATGA 3 ' (SEQ ID NO:47)
Fgf4:5 ' GCGGCTCTACTGCAACGT 3 ' (SEQ ID NO:48) and 5 ' CCTTCTTGGTCTTCCCATTC 3 ' (SEQ ID NO:49)
Gapdh:5 ' AATCCCATCACCATCTTCC3 ' (SEQ ID NO:50) and 5 ' CATCACGCCACAGTTTCC 3 ' (SEQ ID NO:51)
3) formation and the differentiation of embryo's corpusculum (EB):
In order further to estimate the character of the IPS cell that obtains with induction method of the present invention, the existing totipotency that detects the IPS cell that is obtained.Inductive IPS cell is carried out the formation and the differentiation of external evoked embryo's corpusculum (EB), and concrete grammar may further comprise the steps:
(1) takes out the IPS cell that to induce differentiation, abandon substratum, wash one time with PBS;
(2) add Dispase, place 37 ℃ of incubators to digest, digestion time should prolong when going down to posterity to some extent, makes the embryonic stem cell clone come off easily;
(3) take out cell, blow and beat gently, all clones are broken away from the bottom of the culture dish with aseptic glass dropper;
(4) cell mass is transferred in the aseptic 15mL awl end centrifuge tube centrifugal 3 minutes of 1000rpm;
(5) abandon supernatant, the usefulness division culture medium (foetal calf serum 150mL, glutamine 0.146g, beta-mercaptoethanol 50 μ l, non-essential amino acid 10mL is settled to 1000mL with DMEM/F12) resuspended gently;
(6) cell mass is inoculated in (because EB forms easily) in the Petri-dish culture dish that hangs down the attaching ability under condition of suspension culture, puts back in 37 ℃ of incubators;
(7) every other day change a subculture, generally just can see the EB of typical approximate ball sample after 4-5 days, incubation time is decided as required, and the time is long more, and the cell differentiation among the EB is high more.
The observed result of embryo's corpusculum sees that (magnification: * 100), inductive IPS cell all can form embryo's corpusculum (EB) in division culture medium as a result among Fig. 6.After EB forms 7 days, make its adherent continuation differentiation 7 days again, method is: collect and to have cultivated the EB that breaks up behind certain fate, it is inoculated in (the 5ng/ μ l through fibronectin, Sigma) promote in the culture dish of bag quilt that EB's is adherent, still use division culture medium, every other day change once, can observe the differentiated cell types that grows different shape from EB on every side gradually.The result as shown in Figure 6.
Use immunofluorescence method (concrete grammar is with embodiment 5) to detect the gene Nestin of its three germinal layers (ectoderm, mesoderm and entoderm) then, T and AFP gene the results are shown in Figure 7.
By above-mentioned experiment, we have added up above-mentioned inducible factor Oct4 (POU5f1), Sox2, and c-Myc, Klf4, UTF1, the AP positive that Rex1 (ZFP42) and p53si combination is produced, the clone's number that is similar to embryonic stem cell see Table 1 and Fig. 8.
Can produce the exploration of the inducible factor combination of people IPS
In sum, we utilize lentiviral vectors transduction Oct4 (POU5f1), Sox2, and c-Myc, Klf4, UTF1, the full-length cDNA of Rex1 6 genes such as (ZFP42) and the method for p53 gene inhibition agent have realized that the efficient production people induces myeloid-lymphoid stem cell.Can in order to confirm to reach same purpose with the factor still less, we once reject a gene in the combination of seven factors, and add up the AP positive that can produce, the clone's number that is similar to human embryo stem cell, and the result as shown in Figure 8.
For further disclose 3 factors that we find (Klf4, UTF1, Rex1) can substitute original four factors (Oct4, Sox2, c-Myc, function Klf4), we have carried out further experiment according to table 1.Find that Utf1 can substitute the Oct4 functionating, p53 interferes the function that also can substitute Klf4 simultaneously.
Table 1: each inducible factor combination results AP+ and similar embryonic stem cell clone counting statistics table (per 10 5Target cell)
Figure A20081009184100271
Annotate: O (Oct4), S (Sox2), M (c-Myc), K (Klf4), P (P53si), U (Utf1), R (Rex1), the IPS cell clone form that is produced and identify with embodiment 5 Therefore, omited.
Can find out by the form statistics, on the basis of OSMK, add U, R, any one factor among the P all can improve the colony forming efficiency of the AP positive and similar embryonic stem cell, discloses U, R, the P factor is for the plastidogenetic promoter action of IPS; With OSMKPUR is that the basis deducts U respectively, R, and any one among the P all can obviously reduce rate of formation, prompting U, R, P is for the plastidogenetic promoter action of IPS; In addition, P can replace the K that has reported, and U can replace the O that has reported and induce the function that produces the IPS cell with enforcement.
Simultaneously, we have carried out inducing monkey (rhesus monkey, rhesus macaque) to induce the exploration of IPS cell, and the inoblast of promptly getting the monkey ear skin is induced according to the program of the foregoing description to have produced the IPS cell., the IPS cell clone form that is produced and identify with embodiment 5 Therefore, omited.The result: the AP positive colony that 4 factors (OSMK) produce is about 10/20000, do not have similar embryonic stem cell clone, and the AP positive colony that 7 factors (OSMKPUR) are produced is about 200/20000, and similar embryonic stem cell clone is about 20/20000.
Though in concrete implementation process, adopted the inoblast of people or monkey, persons of ordinary skill in the art may appreciate that above-mentioned experiment can be used for Mammals fully, comprise mouse, realize purpose of the present invention.
Reference:
1.Takahashi，K.?&?Yamanaka，S.Induction?of?pluripotent?stem?cells?frommouse?embryonic?and?adult?fibroblast?cultures?by?defined?factors.Cell126，663-76(2006).
2.Okita，K.，Ichisaka，T.?&?Yamanaka，S.Generation?ofgermline-competent?induced?pluripotent?stem?cells.Nature?448，313-7(2007).
3.Wernig，M.et?al.In?vitro?reprogramming?of?fibroblasts?into?apluripotent?ES-cell-like?state.Nature?448，318-24(2007).
4.Maherali，N.et?al.Directly?reprogrammed?fibroblasts?show?globalepigenetic?remodeling?and?widespread?tissue?contribution.Cell?StemCell?1，55-70(2007).
5.Meissner，A.，Wernig，M.?&?Jaenisch，R.Direct?reprogramming?ofgenetically?unmodified?fibroblasts?into?pluripotent?stem?cells.NatBiotechnol?25，1177-81(2007).
6.Blelloch，R.，Venere，M.，Yen，J.?&?Ramalho-Santos，M.Generation?ofinduced?pluripotent?stem?cells?in?the?absence?of?drug?selection.CellStem?Cell?1，245-7(2007).
7.Nakagawa，M.et?al.Generation?of?induced?pluripotent?stem?cellswithout?Myc?from?mouse?and?human?fibroblasts.Nat?Biotechnol?26，101-6(2008).
8.Stadtfeld，M.，Maherali，N.，Breault，D.T.?&?Hochedlinger，K.DefiningMolecular?Cornerstones?during?Fibroblast?to?iPS?Cell?Reprogrammingin?Mouse.Cell?Stem?Cell?2，230-40(2008).
9.Brambrink，T.et?al.Sequential?expression?of?pluripotency?markersduring?direct?reprogramming?of?mouse?somatic?cells.Cell?Stem?Cell?2，151-9(2008).
10.Aoi，T.et?al.Generation?of?Pluripotent?Stem?Cells?from?Adult?MouseLiver?and?Stomach?Cells.Science(2008).
11.Takahashi，K.et?al.Induction?of?pluripotent?stem?cells?from?adulthuman?fibroblasts?by?defined?factors.Cell?131，861-72(2007).
12.Park，I.H.et?al.Reprogramming?of?human?somatic?cells?to?pluripotencywith?defined?factors.Nature?451，141-6(2008).
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14.Qin，H.et?al.Regulation?of?apoptosis?and?differentiation?by?p53?inhuman?embryonic?stem?cells.J?Biol?Chem?282，5842-52(2007).
15.Lin，T.et?al.p53?induces?differentiation?of?mouse?embryonic?stem?cellsby?suppressing?Nanog?expression.Nat?Cell?Biol?7，165-71(2005).
16.Cleland，J.L.，Daugherty，A.?&?Mrsny，R.Emerging?protein?deliverymethods.Curr?Opin?Biotechnol?12，212-9(2001).
SEQUENCE?LISTING
＜110〉Peking University
Company limited of Boke
＜120〉induce method, test kit and the purposes of multipotential cell
<130>IB083130
<160>55
<170>PatentIn?version?3.1
<210>1
<211>55
<212>DNA
＜213〉artificial sequence
<400>1
tgactccagt?ggtaatctac?ttcaagagag?tagattacca?ctggagtctt?ttttc 55
<210>2
<211>59
<212>DNA
＜213〉artificial sequence
<400>2
tcgagaaaaa?agactccagt?ggtaatctac?tctcttgaag?tagattacca?ctggagtca 59
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<400>3
aggatccgcc?accatggcgg?gacacctggc 30
<210>4
<211>30
<212>DNA
＜213〉artificial sequence
<400>4
gcgaattcat?cagtttgaat?gcatgggagg 30
<210>5
<211>1083
<212>DNA
＜213〉people
<400>5
atggcgggac?acctggcttc?agatttcgcc?ttctcgcccc?ctccaggtgg?tggaggtgat 60
gggccagggg?ggccggagcc?gggctgggtt?gatcctcgga?cctggctaag?cttccaaggc 120
cctcctggag?ggccaggaat?cgggccgggg?gttgggccag?gctctgaggt?gtgggggatt 180
cccccatgcc?ccccgccgta?tgagttctgt?ggggggatgg?cgtactgtgg?gccccaggtt 240
ggagtggggc?tagtgcccca?aggcggcttg?gagacctctc?agcctgaggg?cgaagcagga 300
gtcggggtgg?agagcaactc?cgatggggcc?tccccggagc?cctgcaccgt?cacccctggt 360
gccgtgaagc?tggagaagga?gaagctggag?caaaacccgg?aggagtccca?ggacatcaaa 420
gctctgcaga?aagaactcga?gcaatttgcc?aagctcctga?agcagaagag?gatcaccctg 480
ggatatacac?aggccgatgt?ggggctcacc?ctgggggttc?tatttgggaa?ggtattcagc 540
caaacgacca?tctgccgctt?tgaggctctg?cagcttagct?tcaagaacat?gtgtaagctg 600
cggcccttgc?tgcagaagtg?ggtggaggaa?gctgacaaca?atgaaaatct?tcaggagata 660
tgcaaagcag?aaaccctcgt?gcaggcccga?aagagaaagc?gaaccagtat?cgagaaccga 720
gtgagaggca?acctggagaa?tttgttcctg?cagtgcccga?aaccgacact?gcagcagatc 780
agccacatcg?cccagcagct?tgggctcgag?aaggatgtgg?tccgagtgtg?gttctgtaac 840
cggcgccaga?agggcaagcg?atcaagcagc?gactatgcac?aacgagagga?ttttgaggct 900
gctgggtctc?ctttctcagg?gggaccagtg?tcctttcctc?tggccccagg?gccccatttt 960
ggtaccccag?gctatgggag?ccctcacttc?actgcactgt?actcctcggt?ccctttccct 1020
gagggggaag?cctttccccc?tgtctccgtc?accactctgg?gctctcccat?gcattcaaac 1080
tga 1083
<210>6
<211>30
<212>DNA
＜213〉artificial sequence
<400>6
cctcgagcca?ccatgtacaa?catgatggag 30
<210>7
<211>28
<212>DNA
＜213〉artificial sequence
<400>7
cggaattcat?cacatgtgtg?agaggggc 28
<210>8
<211>954
<212>DNA
＜213〉people
<400>8
atgtacaaca?tgatggagac?ggagctgaag?ccgccgggcc?cgcagcaaac?ttcggggggc 60
ggcggcggca?actccaccgc?ggcggcggcc?ggcggcaacc?agaaaaacag?cccggaccgc 120
gtcaagcggc?ccatgaatgc?cttcatggtg?tggtcccgcg?ggcagcggcg?caagatggcc 180
caggagaacc?ccaagatgca?caactcggag?atcagcaagc?gcctgggcgc?cgagtggaaa 240
cttttgtcgg?agacggagaa?gcggccgttc?atcgacgagg?ctaagcggct?gcgagcgctg 300
cacatgaagg?agcacccgga?ttataaatac?cggccccggc?ggaaaaccaa?gacgctcatg 360
aagaaggata?agtacacgct?gcccggcggg?ctgctggccc?ccggcggcaa?tagcatggcg 420
agcggggtcg?gggtgggcgc?cggcctgggc?gcgggcgtga?accagcgcat?ggacagttac 480
gcgcacatga?acggctggag?caacggcagc?tacagcatga?tgcaggacca?gctgggctac 540
ccgcagcacc?cgggcctcaa?tgcgcacggc?gcagcgcaga?tgcagcccat?gcaccgctac 600
gacgtgagcg?ccctgcagta?caactccatg?accagctcgc?agacctacat?gaacggctcg 660
cccacctaca?gcatgtccta?ctcgcagcag?ggcacccctg?gcatggctct?tggctccatg 720
ggttcggtgg?tcaagtccga?ggccagctcc?agcccccctg?tggttacctc?ttcctcccac 780
tccagggcgc?cctgccaggc?cggggacctc?cgggacatga?tcagcatgta?tctccccggc 840
gccgaggtgc?cggaacccgc?cgcccccagc?agacttcaca?tgtcccagca?ctaccagagc 900
ggcccggtgc?ccggcacggc?cattaacggc?acactgcccc?tctcacacat?gtga 954
<210>9
<211>31
<212>DNA
＜213〉artificial sequence
<400>9
tactcgaggc?caccatggct?gtcagcgacg?c 31
<210>10
<211>31
<212>DNA
＜213〉artificial sequence
<400>10
ggcgaattca?ttaaaaatgc?ctcttcatgt?g 31
<210>11
<211>1413
<212>DNA
＜213〉people
<400>11
atggctgtca?gcgacgcgct?gctcccatct?ttctccacgt?tcgcgtctgg?cccggcggga 60
agggagaaga?cactgcgtca?agcaggtgcc?ccgaataacc?gctggcggga?ggagctctcc 120
cacatgaagc?gacttccccc?agtgcttccc?ggccgcccct?atgacctggc?ggcggcgacc 180
gtggccacag?acctggagag?cggcggagcc?ggtgcggctt?gcggcggtag?caacctggcg 240
cccctacctc?ggagagagac?cgaggagttc?aacgatctcc?tggacctgga?ctttattctc 300
tccaattcgc?tgacccatcc?tccggagtca?gtggccgcca?ccgtgtcctc?gtcagcgtca 360
gcctcctctt?cgtcgtcgcc?gtcgagcagc?ggccctgcca?gcgcgccctc?cacctgcagc 420
ttcacctatc?cgatccgggc?cgggaacgac?ccgggcgtgg?cgccgggcgg?cacgggcgga 480
ggcctcctct?atggcaggga?gtccgctccc?cctccgacgg?ctcccttcaa?cctggcggac 540
atcaacgacg?tgagcccctc?gggcggcttc?gtggccgagc?tcctgcggcc?agaattggac 600
ccggtgtaca?ttccgccgca?gcagccgcag?ccgccaggtg?gcgggctgat?gggcaagttc 660
gtgctgaagg?cgtcgctgag?cgcccctggc?agcgagtacg?gcagcccgtc?ggtcatcagc 720
gtcagcaaag?gcagccctga?cggcagccac?ccggtggtgg?tggcgcccta?caacggcggg 780
ccgccgcgca?cgtgccccaa?gatcaagcag?gaggcggtct?cttcgtgcac?ccacttgggc 840
gctggacccc?ctctcagcaa?tggccaccgg?ccggctgcac?acgacttccc?cctggggcgg 900
cagctcccca?gcaggactac?cccgaccctg?ggtcttgagg?aagtgctgag?cagcagggac 960
tgtcaccctg?ccctgccgct?tcctcccggc?ttccatcccc?acccggggcc?caattaccca 1020
tccttcctgc?ccgatcagat?gcagccgcaa?gtcccgccgc?tccattacca?agagctcatg 1080
ccacccggtt?cctgcatgcc?agaggagccc?aagccaaaga?ggggaagacg?atcgtggccc 1140
cggaaaagga?ccgccaccca?cacttgtgat?tacgcgggct?gcggcaaaac?ctacacaaag 1200
agttcccatc?tcaaggcaca?cctgcgaacc?cacacaggtg?agaaacctta?ccactgtgac 1260
tgggacggct?gtggatggaa?attcgcccgc?tcagatgaac?tgaccaggca?ctaccgtaaa 1320
cacacggggc?accgcccgtt?ccagtgccaa?aaatgcgacc?gagcattttc?caggtcggac 1380
cacctcgcct?tacacatgaa?gaggcatttt?taa 1413
<210>12
<211>30
<212>DNA
＜213〉artificial sequence
<400>12
actcgagcca?ccatgcccct?caacgttagc 30
<210>13
<211>30
<212>DNA
＜213〉artificial sequence
<400>13
cggaattcat?tacgcacaag?agttccgtag 30
<210>14
<211>1320
<212>DNA
＜213〉people
<400>14
atgcccctca?acgttagctt?caccaacagg?aactatgacc?tcgactacga?ctcggtgcag 60
ccgtatttct?actgcgacga?ggaggagaac?ttctaccagc?agcagcagca?gagcgagctg 120
cagcccccgg?cgcccagcga?ggatatctgg?aagaaattcg?agctgctgcc?caccccgccc 180
ctgtccccta?gccgccgctc?cgggctctgc?tcgccctcct?acgttgcggt?cacacccttc 240
tcccttcggg?gagacaacga?cggcggtggc?gggagcttct?ccacggccga?ccagctggag 300
atggtgaccg?agctgctggg?aggagacatg?gtgaaccaga?gtttcatctg?cgacccggac 360
gacgagacct?tcatcaaaaa?catcatcatc?caggactgta?tgtggagcgg?cttctcggcc 420
gccgccaagc?tcgtctcaga?gaagctggcc?tcctaccagg?ctgcgcgcaa?agacagcggc 480
agcccgaacc?ccgcccgcgg?ccacagcgtc?tgctccacct?ccagcttgta?cctgcaggat 540
ctgagcgccg?ccgcctcaga?gtgcatcgac?ccctcggtgg?tcttccccta?ccctctcaac 600
gacagcagct?cgcccaagtc?ctgcgcctcg?caagactcca?gcgccttctc?tccgtcctcg 660
gattctctgc?tctcctcgac?ggagtcctcc?ccgcagggca?gccccgagcc?cctggtgctc 720
catgaggaga?caccgcccac?caccagcagc?gactctgagg?aggaacaaga?agatgaggaa 780
gaaatcgatg?ttgtttctgt?ggaaaagagg?caggctcctg?gcaaaaggtc?agagtctgga 840
tcaccttctg?ctggaggcca?cagcaaacct?cctcacagcc?cactggtcct?caagaggtgc 900
cacgtctcca?cacatcagca?caactacgca?gcgcctccct?ccactcggaa?ggactatcct 960
gctgccaaga?gggtcaagtt?ggacagtgtc?agagtcctga?gacagatcag?caacaaccga 1020
aaatgcacca?gccccaggtc?ctcggacacc?gaggagaatg?tcaagaggcg?aacacacaac 1080
gtcttggagc?gccagaggag?gaacgagcta?aaacggagct?tttttgccct?gcgtgaccag 1140
atcccggagt?tggaaaacaa?tgaaaaggcc?cccaaggtag?ttatccttaa?aaaagccaca 1200
gcatacatcc?tgtccgtcca?agcagaggag?caaaagctca?tttctgaaga?ggacttgttg 1260
cggaaacgac?gagaacagtt?gaaacacaaa?cttgaacagc?tacggaactc?ttgtgcgtaa 1320
<210>15
<211>31
<212>DNA
＜213〉artificial sequence
<400>15
aactcgagcc?accatgctgc?tccggccccg?c 31
<210>16
<211>30
<212>DNA
＜213〉artificial sequence
<400>16
aagaattcca?ctggcacggg?tccctgagga 30
<210>17
<211>1026
<212>DNA
＜213〉people
<400>17
atgctgctcc?ggccccgcag?gccgcccccg?ctcgcgcccc?ccgcgccgcc?ctcgcccgcc 60
agccccgacc?ccgagccgcg?gacacccgga?gacgccccgg?ggaccccgcc?ccggaggccc 120
gcctcgccca?gcgcgctggg?ggaactcggg?ttgccggtgt?ccccgggctc?ggcgcagcgc 180
acgccctgga?gcgcccggga?gacggagctg?ctgctgggga?cgctgctgca?accggccgtg 240
tggcgcgcgc?tgctcctgga?ccgccgccag?gccctgccca?cctaccgccg?cgtgtcggcc 300
gcgctggccc?agcagcaggt?gcgccgcacc?cccgcgcagt?gccgccgccg?ctacaagttc 360
cttaaagaca?agtttcgcga?ggcgcacggc?cagccgcccg?ggcccttcga?cgagcagatc 420
cggaagctca?tggggctgct?gggcgacaac?gggcgcaaac?ggcctcgccg?ccgctccccg 480
gggtccgggc?gcccccagcg?cgcccgccgc?ccggtcccca?acgcgcacgc?gccggctccc 540
agcgaaccag?acgccacccc?gctgcccacc?gcccgcgacc?gcgacgcgga?ccccacctgg 600
acgctccgct?tcagcccgtc?cccaccgaag?tctgcggacg?cctcccccgc?ccccggctcc 660
ccgccagctc?ccgccccgac?cgccctcgcc?acctgcatcc?ccgaggaccg?cgcgcccgtc 720
cgcggccccg?ggtccccgcc?gccacccccg?gcccgcgaag?accccgactc?gccgcccggc 780
cgccccgagg?actgcgcgcc?ccctccggcc?gcgcccccgt?cgctgaacac?cgccctgctg 840
cagaccctgg?ggcacctggg?cgacatcgcg?aacatcctgg?gcccgctgcg?cgaccagctg 900
ctgaccttga?accagcacgt?ggagcagctg?cgcggcgcct?tcgaccagac?agtgtccctg 960
gccgtgggct?tcattctggg?cagcgcggcc?gccgagcgag?gggtcctcag?ggacccgtgc 1020
cagtga 1026
<210>18
<211>35
<212>DNA
＜213〉artificial sequence
<400>18
aactcgagcc?accatgagcc?agcaactgaa?gaaac 35
<210>19
<211>30
<212>DNA
＜213〉artificial sequence
<400>19
ttgatatcct?actttccctc?ttgttcattc 30
<210>20
<211>933
<212>DNA
＜213〉people
<400>20
atgagccagc?aactgaagaa?acgggcaaag?acaagacacc?agaaaggcct?gggtggaaga 60
gcccccagtg?gggctaagcc?caggcaaggc?aagtcaagcc?aagacctgca?ggcggaaata 120
gaacctgtca?gcgcggtgtg?ggccttatgt?gatggctatg?tgtgctatga?gcctggccct 180
caggctctcg?gaggggatga?tttctcagac?tgttacatag?aatgcgtcat?aaggggtgag 240
ttttctcaac?ccatcctgga?agaggactca?ctttttgagt?ccttggaata?cctaaagaaa 300
ggatcagaac?aacagctttc?tcaaaaggtt?ttcgaagcaa?gctcccttga?atgttctttg 360
gaatacatga?aaaaaggggt?aaagaaagag?cttccacaaa?agatagttgg?agagaattcg 420
cttgagtatt?ctgagtacat?gacaggcaag?aagcttccgc?ctggaggaat?acctggcatt 480
gacctatcag?atcctaaaca?gctcgcagaa?tttgctagaa?agaagccccc?cataaataaa 540
gaatatgaca?gtctgagcgc?aatcgcttgt?cctcagagtg?gatgcactag?gaagttgagg 600
aatagagctg?ccctgagaaa?gcatctcctc?attcatggtc?cccgagacca?cgtctgtgcg 660
gaatgtggga?aagcgttcgt?tgagagctca?aaactaaaga?gacatttcct?ggttcatact 720
ggagagaagc?cgtttcggtg?cacttttgaa?gggtgcggaa?agcgcttctc?tctggacttt 780
aatttgcgta?cgcacgtgcg?catccacacg?ggggagaaac?gtttcgtgtg?tccctttcaa 840
ggctgcaaca?ggaggtttat?tcagtcaaat?aacctgaaag?cccacatcct?aacgcatgca 900
aatacgaaca?agaatgaaca?agagggaaag?tag 933
<210>21
<211>7650
<212>DNA
＜213〉artificial sequence
<400>21
gtcgacggat?cgggagatct?cccgatcccc?tatggtgcac?tctcagtaca?atctgctctg 60
atgccgcata?gttaagccag?tatctgctcc?ctgcttgtgt?gttggaggtc?gctgagtagt 120
gcgcgagcaa?aatttaagct?acaacaaggc?aaggcttgac?cgacaattgc?atgaagaatc 180
tgcttagggt?taggcgtttt?gcgctgcttc?gcgatgtacg?ggccagatat?acgcgttgac 240
attgattatt?gactagttat?taatagtaat?caattacggg?gtcattagtt?catagcccat 300
atatggagtt?ccgcgttaca?taacttacgg?taaatggccc?gcctggctga?ccgcccaacg 360
acccccgccc?attgacgtca?ataatgacgt?atgttcccat?agtaacgcca?atagggactt 420
tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc?ccacttggca?gtacatcaag 480
tgtatcatat?gccaagtacg?ccccctattg?acgtcaatga?cggtaaatgg?cccgcctggc 540
attatgccca?gtacatgacc?ttatgggact?ttcctacttg?gcagtacatc?tacgtattag 600
tcatcgctat?taccatggtg?atgcggtttt?ggcagtacat?caatgggcgt?ggatagcggt 660
ttgactcacg?gggatttcca?agtctccacc?ccattgacgt?caatgggagt?ttgttttggc 720
accaaaatca?acgggacttt?ccaaaatgtc?gtaacaactc?cgccccattg?acgcaaatgg 780
gcggtaggcg?tgtacggtgg?gaggtctata?taagcagcgc?gttttgcctg?tactgggtct 840
ctctggttag?accagatctg?agcctgggag?ctctctggct?aactagggaa?cccactgctt 900
aagcctcaat?aaagcttgcc?ttgagtgctt?caagtagtgt?gtgcccgtct?gttgtgtgac 960
tctggtaact?agagatccct?cagacccttt?tagtcagtgt?ggaaaatctc?tagcagtggc 1020
gcccgaacag?ggacttgaaa?gcgaaaggga?aaccagagga?gctctctcga?cgcaggactc 1080
ggcttgctga?agcgcgcacg?gcaagaggcg?aggggcggcg?actggtgagt?acgccaaaaa 1140
ttttgactag?cggaggctag?aaggagagag?atgggtgcga?gagcgtcagt?attaagcggg 1200
ggagaattag?atcgcgatgg?gaaaaaattc?ggttaaggcc?agggggaaag?aaaaaatata 1260
aattaaaaca?tatagtatgg?gcaagcaggg?agctagaacg?attcgcagtt?aatcctggcc 1320
tgttagaaac?atcagaaggc?tgtagacaaa?tactgggaca?gctacaacca?tcccttcaga 1380
caggatcaga?agaacttaga?tcattatata?atacagtagc?aaccctctat?tgtgtgcatc 1440
aaaggataga?gataaaagac?accaaggaag?ctttagacaa?gatagaggaa?gagcaaaaca 1500
aaagtaagac?caccgcacag?caagcggccg?gccgcgctga?tcttcagacc?tggaggagga 1560
gatatgaggg?acaattggag?aagtgaatta?tataaatata?aagtagtaaa?aattgaacca 1620
ttaggagtag?cacccaccaa?ggcaaagaga?agagtggtgc?agagagaaaa?aagagcagtg 1680
ggaataggag?ctttgttcct?tgggttcttg?ggagcagcag?gaagcactat?gggcgcagcg 1740
tcaatgacgc?tgacggtaca?ggccagacaa?ttattgtctg?gtatagtgca?gcagcagaac 1800
aatttgctga?gggctattga?ggcgcaacag?catctgttgc?aactcacagt?ctggggcatc 1860
aagcagctcc?aggcaagaat?cctggctgtg?gaaagatacc?taaaggatca?acagctcctg 1920
gggatttggg?gttgctctgg?aaaactcatt?tgcaccactg?ctgtgccttg?gaatgctagt 1980
tggagtaata?aatctctgga?acagatttgg?aatcacacga?cctggatgga?gtgggacaga 2040
gaaattaaca?attacacaag?cttaatacac?tccttaattg?aagaatcgca?aaaccagcaa 2100
gaaaagaatg?aacaagaatt?attggaatta?gataaatggg?caagtttgtg?gaattggttt 2160
aacataacaa?attggctgtg?gtatataaaa?ttattcataa?tgatagtagg?aggcttggta 2220
ggtttaagaa?tagtttttgc?tgtactttct?atagtgaata?gagttaggca?gggatattca 2280
ccattatcgt?ttcagaccca?cctcccaacc?ccgaggggac?ccgacaggcc?cgaaggaata 2340
gaagaagaag?gtggagagag?agacagagac?agatccattc?gattagtgaa?cggatcggca 2400
ctgcgtgcgc?caattctgca?gacaaatggc?agtattcatc?cacaatttta?aaagaaaagg 2460
ggggattggg?gggtacagtg?caggggaaag?aatagtagac?ataatagcaa?cagacataca 2520
aactaaagaa?ttacaaaaac?aaattacaaa?aattcaaaat?tttcgggttt?attacaggga 2580
cagcagagat?ccagtttggt?tagtaccggg?cccgctctag?agatccgacg?ccgccatctc 2640
taggcccgcg?ccggccccct?cgcacagact?tgtgggagaa?gctcggctac?tcccctgccc 2700
cggttaattt?gcatataata?tttcctagta?actatagagg?cttaatgtgc?gataaaagac 2760
agataatctg?ttctttttaa?tactagctac?attttacatg?ataggcttgg?atttctataa 2820
gagatacaaa?tactaaatta?ttattttaaa?aaacagcaca?aaaggaaact?caccctaact 2880
gtaaagtaat?tgtgtgtttt?gagactataa?atatcccttg?gagaaaagcc?ttgttaacgc 2940
gcggtgaccc?tcgaggtcga?cggtatcgat?aagctcgctt?cacgagattc?cagcaggtcg 3000
agggacctaa?taacttcgta?tagcatacat?tatacgaagt?tatattaagg?gttccaagct 3060
taagcggccg?cgtggataac?cgtattaccg?ccatgcatta?gttattaata?gtaatcaatt 3120
acggggtcat?tagttcatag?cccatatatg?gagttccgcg?ttacataact?tacggtaaat 3180
ggcccgcctg?gctgaccgcc?caacgacccc?cgcccattga?cgtcaataat?gacgtatgtt 3240
cccatagtaa?cgccaatagg?gactttccat?tgacgtcaat?gggtggagta?tttacggtaa 3300
actgcccact?tggcagtaca?tcaagtgtat?catatgccaa?gtacgccccc?tattgacgtc 3360
aatgacggta?aatggcccgc?ctggcattat?gcccagtaca?tgaccttatg?ggactttcct 3420
acttggcagt?acatctacgt?attagtcatc?gctattacca?tggtgatgcg?gttttggcag 3480
tacatcaatg?ggcgtggata?gcggtttgac?tcacggggat?ttccaagtct?ccaccccatt 3540
gacgtcaatg?ggagtttgtt?ttggcaccaa?aatcaacggg?actttccaaa?atgtcgtaac 3600
aactccgccc?cattgacgca?aatgggcggt?aggcgtgtac?ggtgggaggt?ctatataagc 3660
agagctggtt?tagtgaaccg?tcagatccgc?tagcgctacc?ggtcgccacc?atggtgagca 3720
agggcgagga?gctgttcacc?ggggtggtgc?ccatcctggt?cgagctggac?ggcgacgtaa 3780
acggccacaa?gttcagcgtg?tccggcgagg?gcgagggcga?tgccacctac?ggcaagctga 3840
ccctgaagtt?catctgcacc?accggcaagc?tgcccgtgcc?ctggcccacc?ctcgtgacca 3900
ccctgaccta?cggcgtgcag?tgcttcagcc?gctaccccga?ccacatgaag?cagcacgact 3960
tcttcaagtc?cgccatgccc?gaaggctacg?tccaggagcg?caccatcttc?ttcaaggacg 4020
acggcaacta?caagacccgc?gccgaggtga?agttcgaggg?cgacaccctg?gtgaaccgca 4080
tcgagctgaa?gggcatcgac?ttcaaggagg?acggcaacat?cctggggcac?aagctggagt 4140
acaactacaa?cagccacaac?gtctatatca?tggccgacaa?gcagaagaac?ggcatcaagg 4200
tgaacttcaa?gatccgccac?aacatcgagg?acggcagcgt?gcagctcgcc?gaccactacc 4260
agcagaacac?ccccatcggc?gacggccccg?tgctgctgcc?cgacaaccac?tacctgagca 4320
cccagtccgc?cctgagcaaa?gaccccaacg?agaagcgcga?tcacatggtc?ctgctggagt 4380
tcgtgaccgc?cgccgggatc?actctcggca?tggacgagct?gtacaagtag?gaattcgtcg 4440
agggacctaa?taacttcgta?tagcatacat?tatacgaagt?tatacatgtt?taagggttcc 4500
ggttccacta?ggtacaattc?gatatcaagc?ttatcgataa?tcaacctctg?gattacaaaa 4560
tttgtgaaag?attgactggt?attcttaact?atgttgctcc?ttttacgcta?tgtggatacg 4620
ctgctttaat?gcctttgtat?catgctattg?cttcccgtat?ggctttcatt?ttctcctcct 4680
tgtataaatc?ctggttgctg?tctctttatg?aggagttgtg?gcccgttgtc?aggcaacgtg 4740
gcgtggtgtg?cactgtgttt?gctgacgcaa?cccccactgg?ttggggcatt?gccaccacct 4800
gtcagctcct?ttccgggact?ttcgctttcc?ccctccctat?tgccacggcg?gaactcatcg 4860
ccgcctgcct?tgcccgctgc?tggacagggg?ctcggctgtt?gggcactgac?aattccgtgg 4920
tgttgtcggg?gaaatcatcg?tcctttcctt?ggctgctcgc?ctgtgttgcc?acctggattc 4980
tgcgcgggac?gtccttctgc?tacgtccctt?cggccctcaa?tccagcggac?cttccttccc 5040
gcggcctgct?gccggctctg?cggcctcttc?cgcgtcttcg?ccttcgccct?cagacgagtc 5100
ggatctccct?ttgggccgcc?tccccgcatc?gataccgtcg?acctcgatcg?agacctagaa 5160
aaacatggag?caatcacaag?tagcaataca?gcagctacca?atgctgattg?tgcctggcta 5220
gaagcacaag?aggaggagga?ggtgggtttt?ccagtcacac?ctcaggtacc?tttaagacca 5280
atgacttaca?aggcagctgt?agatcttagc?cactttttaa?aagaaaaggg?gggactggaa 5340
gggctaattc?actcccaacg?aagacaagat?atccttgatc?tgtggatcta?ccacacacaa 5400
ggctacttcc?ctgattggca?gaactacaca?ccagggccag?ggatcagata?tccactgacc 5460
tttggatggt?gctacaagct?agtaccagtt?gagcaagaga?aggtagaaga?agccaatgaa 5520
ggagagaaca?cccgcttgtt?acaccctgtg?agcctgcatg?ggatggatga?cccggagaga 5580
gaagtattag?agtggaggtt?tgacagccgc?ctagcatttc?atcacatggc?ccgagagctg 5640
catccggact?gtactgggtc?tctctggtta?gaccagatct?gagcctggga?gctctctggc 5700
taactaggga?acccactgct?taagcctcaa?taaagcttgc?cttgagtgct?tcaagtagtg 5760
tgtgcccgtc?tgttgtgtga?ctctggtaac?tagagatccc?tcagaccctt?ttagtcagtg 5820
tggaaaatct?ctagcagcat?gtgagcaaaa?ggccagcaaa?aggccaggaa?ccgtaaaaag 5880
gccgcgttgc?tggcgttttt?ccataggctc?cgcccccctg?acgagcatca?caaaaatcga 5940
cgctcaagtc?agaggtggcg?aaacccgaca?ggactataaa?gataccaggc?gtttccccct 6000
ggaagctccc?tcgtgcgctc?tcctgttccg?accctgccgc?ttaccggata?cctgtccgcc 6060
tttctccctt?cgggaagcgt?ggcgctttct?catagctcac?gctgtaggta?tctcagttcg 6120
gtgtaggtcg?ttcgctccaa?gctgggctgt?gtgcacgaac?cccccgttca?gcccgaccgc 6180
tgcgccttat?ccggtaacta?tcgtcttgag?tccaacccgg?taagacacga?cttatcgcca 6240
ctggcagcag?ccactggtaa?caggattagc?agagcgaggt?atgtaggcgg?tgctacagag 6300
ttcttgaagt?ggtggcctaa?ctacggctac?actagaagaa?cagtatttgg?tatctgcgct 6360
ctgctgaagc?cagttacctt?cggaaaaaga?gttggtagct?cttgatccgg?caaacaaacc 6420
accgctggta?gcggtggttt?ttttgtttgc?aagcagcaga?ttacgcgcag?aaaaaaagga 6480
tctcaagaag?atcctttgat?cttttctacg?gggtctgacg?ctcagtggaa?cgaaaactca 6540
cgttaaggga?ttttggtcat?gagattatca?aaaaggatct?tcacctagat?ccttttaaat 6600
taaaaatgaa?gttttaaatc?aatctaaagt?atatatgagt?aaacttggtc?tgacagttac 6660
caatgcttaa?tcagtgaggc?acctatctca?gcgatctgtc?tatttcgttc?atccatagtt 6720
gcctgactcc?ccgtcgtgta?gataactacg?atacgggagg?gcttaccatc?tggccccagt 6780
gctgcaatga?taccgcgaga?cccacgctca?ccggctccag?atttatcagc?aataaaccag 6840
ccagccggaa?gggccgagcg?cagaagtggt?cctgcaactt?tatccgcctc?catccagtct 6900
attaattgtt?gccgggaagc?tagagtaagt?agttcgccag?ttaatagttt?gcgcaacgtt 6960
gttgccattg?ctacaggcat?cgtggtgtca?cgctcgtcgt?ttggtatggc?ttcattcagc 7020
tccggttccc?aacgatcaag?gcgagttaca?tgatccccca?tgttgtgcaa?aaaagcggtt 7080
agctccttcg?gtcctccgat?cgttgtcaga?agtaagttgg?ccgcagtgtt?atcactcatg 7140
gttatggcag?cactgcataa?ttctcttact?gtcatgccat?ccgtaagatg?cttttctgtg 7200
actggtgagt?actcaaccaa?gtcattctga?gaatagtgta?tgcggcgacc?gagttgctct 7260
tgcccggcgt?caatacggga?taataccgcg?ccacatagca?gaactttaaa?agtgctcatc 7320
attggaaaac?gttcttcggg?gcgaaaactc?tcaaggatct?taccgctgtt?gagatccagt 7380
tcgatgtaac?ccactcgtgc?acccaactga?tcttcagcat?cttttacttt?caccagcgtt 7440
tctgggtgag?caaaaacagg?aaggcaaaat?gccgcaaaaa?agggaataag?ggcgacacgg 7500
aaatgttgaa?tactcatact?cttccttttt?caatattatt?gaagcattta?tcagggttat 7560
tgtctcatga?gcggatacat?atttgaatgt?atttagaaaa?ataaacaaat?aggggttccg 7620
cgcacatttc?cccgaaaagt?gccacctgac 7650
<210>22
<211>20
<212>DNA
＜213〉artificial sequence
<400>22
gaaccgagtg?agaggcaacc 20
<210>23
<211>20
<212>DNA
＜213〉artificial sequence
<400>23
atcccaaaaa?ccctggcaca 20
<210>24
<211>20
<212>DNA
＜213〉artificial sequence
<400>24
atgggttcgg?tggtcaagtc 20
<210>25
<211>20
<212>DNA
＜213〉artificial sequence
<400>25
ccctcccatt?tccctcgttt 20
<210>26
<211>20
<212>DNA
＜213〉artificial sequence
<400>26
tggaacagtc?ccttctataa 20
<210>27
<211>20
<212>DNA
＜213〉artificial sequence
<400>27
ctgattaggc?tccaaccata 20
<210>28
<211>18
<212>DNA
＜213〉artificial sequence
<400>28
accactgtga?ctgggacg 18
<210>29
<211>20
<212>DNA
＜213〉artificial sequence
<400>29
gcagcgtatc?cacatagcgt 20
<210>30
<211>20
<212>DNA
＜213〉artificial sequence
<400>30
tacatcctgt?ccgtccaagc 20
<210>31
<211>20
<212>DNA
＜213〉artificial sequence
<400>31
gcagcgtatc?cacatagcgt 20
<210>32
<211>19
<212>DNA
＜213〉artificial sequence
<400>32
tcattcatgg?tccccgaga 19
<210>33
<211>20
<212>DNA
＜213〉artificial sequence
<400>33
gcagcgtatc?cacatagcgt 20
<210>34
<211>19
<212>DNA
＜213〉artificial sequence
<400>34
gaccagctgc?tgaccttga 19
<210>35
<211>20
<212>DNA
＜213〉artificial sequence
<400>35
gcagcgtatc?cacatagcgt 20
<210>36
<211>26
<212>DNA
＜213〉artificial sequence
<400>36
cagccaagtc?tgtgacttgc?acgtac 26
<210>37
<211>26
<212>DNA
＜213〉artificial sequence
<400>37
ctatgtcgaa?aagtgtttct?gtcatc 26
<210>38
<211>18
<212>DNA
＜213〉artificial sequence
<400>38
gggcatctgt?aagtggtt 18
<210>39
<211>18
<212>DNA
＜213〉artificial sequence
<400>39
gtagggctgt?ggatttct 18
<210>40
<211>18
<212>DNA
＜213〉artificial sequence
<400>40
cccgagactt?atgctacg 18
<210>41
<211>18
<212>DNA
＜213〉artificial sequence
<400>41
tccaggaata?acccgaaa 18
<210>42
<211>18
<212>DNA
＜213〉artificial sequence
<400>42
aaaccctcaa?cttgctcc 18
<210>43
<211>18
<212>DNA
＜213〉artificial sequence
<400>43
ttggtcttcg?gcctctat 18
<210>44
<211>18
<212>DNA
＜213〉artificial sequence
<400>44
tcaggaattt?gctcgtcc 18
<210>45
<211>17
<212>DNA
＜213〉artificial sequence
<400>45
cttgggcagc?caggtgt 17
<210>46
<211>18
<212>DNA
＜213〉artificial sequence
<400>46
atctgggcta?agtgctgg 18
<210>47
<211>18
<212>DNA
＜213〉artificial sequence
<400>47
aagctgggtc?ttggatga 18
<210>48
<211>18
<212>DNA
＜213〉artificial sequence
<400>48
gcggctctac?tgcaacgt 18
<210>49
<211>20
<212>DNA
＜213〉artificial sequence
<400>49
ccttcttggt?cttcccattc 20
<210>50
<211>19
<212>DNA
＜213〉artificial sequence
<400>50
aatcccatca?ccatcttcc 19
<210>51
<211>18
<212>DNA
＜213〉artificial sequence
<400>51
catcacgcca?cagtttcc 18
<210>52
<211>27
<212>DNA
＜213〉artificial sequence
<400>52
gtaggatcca?tgaccgagta?caagccc 27
<210>53
<211>25
<212>DNA
＜213〉artificial sequence
<400>53
gatgaattca?ggcaccgggc?ttgcg 25
<210>54
<211>23
<212>DNA
＜213〉artificial sequence
<400>54
gacuccagug?guaaucuacd?tdt 23
<210>55
<211>23
<212>DNA
＜213〉artificial sequence
<400>55
guagauuacc?acuggagucd?tdt 23

Claims (18)

1. inducible factor UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a is used to the purposes of inducing pluripotent stem cell to produce.
2. inducible factor UTF1, at least a purposes that is used to promote to induce the efficient that produces multipotent stem cells of Rex1 (ZFP42) and the agent of p53 gene inhibition.
3. one kind prepares the method for inducing pluripotent stem cell, and comprising in the cell of differentiation provides inducible factor, and described inducible factor comprises Oct4 (POU5f1), Sox2, c-Myc and Klf4, and UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a.
4. the method for claim 3, wherein said inducible factor does not comprise Oct4 (POU5f1), Sox2, a kind of among c-Myc and the Klf4.
5. the method for claim 4 does not comprise Oct4 when wherein inducible factor comprises UTF1; When comprising p53si, inducible factor do not comprise KLF4.
6. the method for claim 5, wherein inducible factor does not comprise c-Myc.
7. the method for claim 6, the cell of wherein said differentiation is an inoblast, liver cell, gastric cells, keratinocyte or blood cell.
8. the method for claim 7, the cell of wherein said differentiation derives from Mammals.
9. the method for claim 8, wherein said Mammals is people, mouse or primate.
10. each method of claim 3-9, wherein said inducible factor is a dna form, mRNA form or protein form.
11 1 kinds prepare the test kit of inducing pluripotent stem cell, and comprising in the cell of differentiation provides inducible factor, and described inducible factor comprises Oct4 (POU5f1), Sox2, c-Myc and Klf4, and UTF1, Rex1 (ZFP42) and the agent of p53 gene inhibition at least a.
12. the test kit of claim 11, wherein inducible factor does not comprise Oct4 (POU5f1), Sox2, a kind of among c-Myc and the Klf4.
13. the test kit of claim 12 does not comprise Oct4 when wherein inducible factor is UTF1; When being p53si, inducible factor do not comprise KLF4.
14. the test kit of claim 13, wherein said inducible factor does not comprise c-Myc.
15. each test kit of claim 11-14, wherein said inducible factor is a dna form, mRNA form or protein form.
16. each test kit of claim 11-14, wherein when described inducible factor was dna form, described test kit also comprised and being used for the virus vector of inducible factor transfection to noble cells.
17. each test kit of claim 11-14, wherein said virus vector is selected from lentiviral vectors, retroviral vector or adenovirus carrier.
18. each method or each the pluripotent stem cell of test kit preparation of claim 11-17 of claim 3-10.
CN200810091841.2A 2008-04-03 2008-04-03 Prepare the method for pluripotent stem cell, test kit and purposes Expired - Fee Related CN101550406B (en)

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